Substrate specificity of human recombinant mitochondrial deoxyguanosine kinase with cytostatic and antiviral purine and pyrimidine analogs.
Deoxyguanosine kinase (dGK) is an enzyme responsible for the phosphorylation of purine deoxynucleosides in mitochondria of mammalian cells. Its role in activation of pharmacologically used nucleoside analogs is not well understood, because of the low levels of dGK found in tissue extracts and its inactivation during purification. The cDNA for dGK was recently cloned and expressed in Escherichia coli. Here we present an improved procedure for expression and purification of a highly active form of human recombinant dGK. The enzyme showed a broad substrate specificity toward natural purine and pyrimidine deoxynucleosides as well as toward important nucleoside analogs. The Km and Vmax values for deoxyguanosine, deoxyinosine, deoxyadenosine, and deoxycytidine were 4, 13, 460, 330 microM and 43, 330, 430 and 60 nmol/min/mg of protein, respectively. Antileukemic purine analogs such as arabinosyl guanine, 2-chloro-2'-deoxyadenosine, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, and 2-fluoro-arabinosyl-adenine were phosphorylated as efficiently by dGK as the natural nucleoside substrates. This is the first report in which 2-fluoro-arabinosyl-adenine and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine were shown to be good substrates for dGK. The antiviral analogs dideoxyinosine and arabinosyl adenine also showed significant activity with dGK, as did several pyrimidine analogs (e.g., the cytostatic drugs 5-fluoro-2'-deoxycytidine and difluorodeoxycytidine). The broad specificity of dGK described here may change our understanding of the mechanisms responsible for the efficacy and mitochondrial toxicity of several nucleoside analogs.
Sjöberg
A H
AH
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden.
Wang
L
L
Eriksson
S
S
D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011684:Purine Nucleosides / Q000378:metabolism*; D011741:Pyrimidine Nucleosides / Q000378:metabolism*; D011994:Recombinant Proteins; D013379:Substrate Specificity
D011684:Purine Nucleosides; D011741:Pyrimidine Nucleosides; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C019165:deoxyguanosine kinase